Project Summary: Both coding and non-coding DNA genomic sequences as well as their functional significance are becoming increasingly available. Modulating the functions of these sequences with sequence-specific, cell-permeable synthetic compounds would be extremely valuable. Deficiency of satisfactory synthetic compounds is a barrier to applications of our genomic knowledge in biotechnology and drug development. The project described herein will remove that barrier and will have an impact on human health through the potential for new anticancer and antiparasitic drugs as well as new biotechnology applications. The project builds on very successful results in our initial funding period. We found that it is possible to rationally design and prepare novel modules for both AT and GC base pair (BP) recognition in target DNA sequences. For the first time we have designed several new heterocyclic, cationic modules which selectively and strongly recognize a single GC bp with flanking AT sequences. This was a primary goal of our initial proposal and what we propose here builds from that success. Our design research was initiated with classical types of cell permeable AT specific minor groove binders that are based on a molecular platform that includes clinically useful compounds. Our target compounds maintain these features while incorporating new GC recognition modules. The downfall of most minor groove binders in therapeutics has been lack of sufficient cell and nuclear permeability. Our new compounds escaped this block but since we wish to target diseases from those induced by cancer to microorganisms, in Aim 1 we will continue to design, prepare and test new sequence-specific modules for cell permeability and biological activity. Aim 2 of the proposal describes the preparation of entirely new types of mixed sequence recognition compounds with our established modules from Aim 1 linked by both serial and parallel methods to bind tightly and specifically to a broad array of mixed DNA sequences. Preliminary biophysical findings from our first funding period are a proof of concept that our linked modular design approach works and can be expanded to more complex sequences in the next funding period. A broad array of biophysical studies are performed on the new compound-DNA complexes including high resolution NMR methods and crystallography. Aim 3 is entirely new and was not a part of our initial proposal. It builds on exciting preliminary results that illustrate important biological functions of our designed compounds. As a test system, collaborative results with the PU.1 transcription factor (TF) showed that new minor groove agents, identified in our biosensor in vitro assay, enter cells and nuclei and allosterically inhibit major groove binding TFs. This important result for future development of an array of TF inhibitors lead to collaborative evaluation of our compounds against low PU.1 acute myeloid leukemia (AML) patient cells. The compounds entered the cell and nuclei with selective PU.1 inhibition and AML cell death with no toxicity to cells to normal PU.1 cells. These results are the basis for Aim 3 with significant impact and major relevance for new drug development.